Genome modification technologies have been used for decades to study gene function. Over the years, the field has progressively developed tools that enable greater and greater target specificity. Transposable elements offered one of the first means of stably altering genome structure, but their target site specificity is typically poor, allowing them to integrate at many different locations within a single genome. To achieve greater targeting accuracy and fewer targeting events, “targeted nucleases” were developed. Early targeted nucleases were formed by fusing the site-specific DNA-binding domains of transcription factors with the endonuclease domains of restriction enzymes. Examples of such targeted nucleases include the Zinc Finger Nucleases (ZFNs) and Transcriptional Activator-Like Effector Nucleases (TALENS). By changing the DNA-binding domain, the target specificity of such targeted nucleases could be selectively modified.
A third, most recently developed technology is based on the prokaryotic Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) system. Found in bacteria and archaea, CRISPR/Cas functions as an adaptive immune system in which a “programmable endonuclease” associates with small RNAs derived from CRISPR transcripts. The small RNAs direct the programmable endonuclease to complementary DNA sequences, typically found in infectious agents such as bacteriophage, which are then cleaved. To be cleaved, a target site must not only be complementary to the CRISPR RNA, but also must be positioned adjacent to a short sequence motif (the Protospacer Adjacent Motif or “PAM”) that is recognized by the programmable endonuclease. In 2013, it was discovered that a simplified version of the CRISPR/Cas system consisting of a Cas9 endonuclease and a single RNA molecule (referred to as a guide RNA or “gRNA”) could be used to induce targeted nucleolytic cleavage of endogenous genomic sites in mammalian cells. Cong et al. (2013), Multiplex genome engineering using CRISPR/Cas systems, Science 339, 819-823; Mali et al. (2013), RNA-guided human genome engineering via Cas9, Science 339, 823-826; Jinek et al. (2013), RNA-programmed genome editing in human cells, eLife 2, e00471. The relative ease of programming the Cas9 nuclease with short gRNAs specifically targeted to individual loci has led to its rapid adoption as the method of choice for genome editing.
Following nucleolytic cleavage by targeted endonucleases, endogenous cellular DNA repair pathways are activated. DNA breaks are generally repaired via one of two major pathways referred to as non-homologous end-joining (NHEJ) and homology-directed repair (HDR). In NHEJ, double-strand breaks are repaired by religation of the cleaved ends without the involvement of any additional donor or template DNA. This repair pathway is error prone, resulting in insertions and/or deletions (indels) of various sizes at the site of the break. Thus, the most straightforward form of gene editing relies upon indel-mediated disruption of gene function; for example, the introduction of frameshift mutations in the coding sequence of a gene. HDR-mediated mechanisms afford the opportunity to edit genomic sites in a more precise manner by providing donor DNA as a template for repair. While the sister chromosome is naturally available to serve as the donor, exogenously introduced DNA can function as a donor template, particularly in the context of an induced double-strand break. The exogenous donor template allows for the replacement of endogenous nucleotides with any desired sequence. Using this approach, a mutant gene can be converted to its wild-type counterpart, or vice versa.
Although genome modification/editing technologies have become excellent tools for introducing precise, targeted alterations in a genome, the problems of NHEJ and off-target modifications still exist. Moreover, identifying NHEJ and off-target modifications can be costly and time consuming. The present disclosure addresses, among other things, these and other related problems that exist in current genome modification/editing technologies.